HIV-1 Gag is translated in the cytoplasm and trafficked to the plasma membrane, where it assembles into spherical particles that bud from the cell. Proposed studies will address the structural biology, biochemistry,and molecular virology of viral membrane targeting and budding. The first aim is to determine the solution structure, dynamics, and membrane biochemistry of the myristoylated Gag MA domain. These studies, together with our previous structure of myristoylated MA, should reveal the molecular mechanism of the "myristoyl switch" that governs Gag targeting to the plasma membrane. The second aim is to analyze the biochemistry of virus budding. We have identified human Tsg101 as an attractive candidate for the cellular factor that binds the Gag p6 "Late" domain and facilitates virus release. Tsg101 appears to coordinate the cellular pathways of endocytosis, exocytosis, and vacuolar protein sorting (Vps), suggesting that machinery from these pathways may be recruited to assist in the viral release. We will now fully characterize the network of cellular proteins that define the different Tsg101 pathways. These studies will employ high throughput approaches for quantitating putative protein-protein interactions initially identified in automated two-hybrid screens. The third aim is to determine the solution structure of the N-terminal, p6 binding domain of Tsg101, both free and in complex with its p6 binding site. Our preliminary NMR and biochemical analyses indicate that this domain is structurally similar to E2 enzymes that function in the transfer of ubiquitin (denoted Tsg101 E2*) and that peptides spanning the "PTAP" sequence motif of ID:V-1 p6 bind specifically to a site surrounding Tsg101 E2* beta-strand 4. The structure of Tsg101 in complex with its p6 binding site should reveal how the virus can recruit Tsg101 to assist in virus budding and may serve as the basis for structure-based design of inhibitors of viral egress. The final aim is to investigate the molecular virology of HIV-1 budding. We have demonstrated that dominant negative constructs of Vps4, which mislocalize Tsg101 and inhibit vacuolar protein sorting, also block HIV-1 release. We will now characterize the functional role of Tsg101 and the Vps pathway in the viral budding process.